The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.
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We thank L. Looger and colleagues for sharing their unpublished acetylcholine sensors that validated some of our results. We thank Y. Rao for generous sharing of two-photon microscopy. We are also grateful to L. Luo, S. Owen, Y. Rao, and L. Nevin for critical reading of the manuscript. We thank Z. Ye for the help in art designing. This work was supported by the National Basic Research Program of China (973 Program; grant 2015CB856402), The General Program of National Natural Science Foundation of China (project 31671118 and project 31371442), and the Junior Thousand Talents Program of China to Y.L. Additional support comes from NIH grants NS103558 (Y.L. and L.I.Z.), DC008983 (L.I.Z.), MH104227 and MH109475 (Y.Z.), MH109104 and NS022061 (L.W.R.), LH089717 (P.Q.B.), and NS053570, NS091452, NS094980, NS092548, and NS104670 (J.J.Z.). J.J.Z. is the Radboud Professor and Sir Yue-Kong Pao Chair Professor.
M.J. and Y.L. have filed patent applications whose value might be affected by this publication.
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Jing, M., Zhang, P., Wang, G. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat Biotechnol 36, 726–737 (2018). https://doi.org/10.1038/nbt.4184
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